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Auto Tag: Genome assembly

July 19, 2019

Long-read sequencing and de novo genome assembly of Ammopiptanthus nanus, a desert shrub.

Ammopiptanthus nanus is a rare broad-leaved shrub that is found in the desert and arid regions of Central Asia. This plant species exhibits extremely high tolerance to drought and freezing and has been used in abiotic tolerance research in plants. As a relic of the tertiary period, A. nanus is of great significance to plant biogeographic research in the ancient Mediterranean region. Here, we report a draft genome assembly using the Pacific Biosciences (PacBio) platform and gene annotation for A. nanus.A total of 64.72 Gb of raw PacBio sequel reads were generated from four 20-kb libraries. After filtering, 64.53 Gb of clean reads were obtained, giving 72.59× coverage depth. Assembly using Canu gave an assembly length of 823.74 Mb, with a contig N50 of 2.76 Mb. The final size of the assembled A. nanus genome was close to the 889 Mb estimated by k-mer analysis. The gene annotation completeness was evaluated using Benchmarking Universal Single-Copy Orthologs; 1,327 of the 1,440 conserved genes (92.15%) could be found in the A. nanus assembly. Genome annotation revealed that 74.08% of the A. nanus genome is composed of repetitive elements and 53.44% is composed of long terminal repeat elements. We predicted ?37,188 protein-coding genes, of which 96.53% were functionally annotated.The genomic sequences of A. nanus could be a valuable source for comparative genomic analysis in the legume family and will be useful for understanding the phylogenetic relationships of the Thermopsideae and the evolutionary response of plant species to the Qinghai Tibetan Plateau uplift.

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July 19, 2019

Comparison between complete genomes of an isolate of Pseudomonas syringae pv. actinidiae from Japan and a New Zealand isolate of the pandemic.

The modern pandemic of the bacterial kiwifruit pathogen Pseudomonas syringae pv actinidiae (Psa) is caused by a particular Psa lineage. To better understand the genetic basis of the virulence of this lineage, we compare the completely assembled genome of a pandemic New Zealand strain with that of the Psa type strain first isolated in Japan in 1983. Aligning the two genomes shows numerous translocations, constrained so as to retain the appropriate orientation of the Architecture Imparting Sequences (AIMs). There are several large horizontally acquired regions, some of which include Type I, Type II or Type III restriction systems. The activity of these systems is reflected in the methylation patterns of the two strains. The pandemic strain carries an Integrative Conjugative Element (ICE) located at a tRNA-Lys site. Two other complex elements are also present at tRNA-Lys sites in the genome. These elements are derived from ICE but have now acquired some alternative secretion function. There are numerous types of mobile element in the two genomes. Analysis of these elements reveals no evidence of recombination between the two Psa lineages.

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July 19, 2019

Adaptation and conservation insights from the koala genome.

The koala, the only extant species of the marsupial family Phascolarctidae, is classified as ‘vulnerable’ due to habitat loss and widespread disease. We sequenced the koala genome, producing a complete and contiguous marsupial reference genome, including centromeres. We reveal that the koala’s ability to detoxify eucalypt foliage may be due to expansions within a cytochrome P450 gene family, and its ability to smell, taste and moderate ingestion of plant secondary metabolites may be due to expansions in the vomeronasal and taste receptors. We characterized novel lactation proteins that protect young in the pouch and annotated immune genes important for response to chlamydial disease. Historical demography showed a substantial population crash coincident with the decline of Australian megafauna, while contemporary populations had biogeographic boundaries and increased inbreeding in populations affected by historic translocations. We identified genetically diverse populations that require habitat corridors and instituting of translocation programs to aid the koala’s survival in the wild.

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July 19, 2019

Surfing the genomic new wave.

In the last decade, high-throughput sequencing approaches have revolutionized the field of plant genomics. With the pace of technical improvement showing no sign of slowing what advances could be just around the corner.

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July 19, 2019

Fern genomes elucidate land plant evolution and cyanobacterial symbioses.

Ferns are the closest sister group to all seed plants, yet little is known about their genomes other than that they are generally colossal. Here, we report on the genomes of Azolla filiculoides and Salvinia cucullata (Salviniales) and present evidence for episodic whole-genome duplication in ferns-one at the base of ‘core leptosporangiates’ and one specific to Azolla. One fern-specific gene that we identified, recently shown to confer high insect resistance, seems to have been derived from bacteria through horizontal gene transfer. Azolla coexists in a unique symbiosis with N2-fixing cyanobacteria, and we demonstrate a clear pattern of cospeciation between the two partners. Furthermore, the Azolla genome lacks genes that are common to arbuscular mycorrhizal and root nodule symbioses, and we identify several putative transporter genes specific to Azolla-cyanobacterial symbiosis. These genomic resources will help in exploring the biotechnological potential of Azolla and address fundamental questions in the evolution of plant life.

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July 19, 2019

Population genomics shows no distinction between pathogenic Candida krusei and environmental Pichia kudriavzevii: One species, four names.

We investigated genomic diversity of a yeast species that is both an opportunistic pathogen and an important industrial yeast. Under the name Candida krusei, it is responsible for about 2% of yeast infections caused by Candida species in humans. Bloodstream infections with C. krusei are problematic because most isolates are fluconazole-resistant. Under the names Pichia kudriavzevii, Issatchenkia orientalis and Candida glycerinogenes, the same yeast, including genetically modified strains, is used for industrial-scale production of glycerol and succinate. It is also used to make some fermented foods. Here, we sequenced the type strains of C. krusei (CBS573T) and P. kudriavzevii (CBS5147T), as well as 30 other clinical and environmental isolates. Our results show conclusively that they are the same species, with collinear genomes 99.6% identical in DNA sequence. Phylogenetic analysis of SNPs does not segregate clinical and environmental isolates into separate clades, suggesting that C. krusei infections are frequently acquired from the environment. Reduced resistance of strains to fluconazole correlates with the presence of one gene instead of two at the ABC11-ABC1 tandem locus. Most isolates are diploid, but one-quarter are triploid. Loss of heterozygosity is common, including at the mating-type locus. Our PacBio/Illumina assembly of the 10.8 Mb CBS573T genome is resolved into 5 complete chromosomes, and was annotated using RNAseq support. Each of the 5 centromeres is a 35 kb gene desert containing a large inverted repeat. This species is a member of the genus Pichia and family Pichiaceae (the methylotrophic yeasts clade), and so is only distantly related to other pathogenic Candida species.

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July 19, 2019

A high-quality, long-read de novo genome assembly to aid conservation of Hawaii’s last remaining crow species

Genome-level data can provide researchers with unprecedented precision to examine the causes and genetic consequences of population declines, which can inform conservation management. Here, we present a high-quality, long-read, de novo genome assembly for one of the world’s most endangered bird species, the ?Alala (Corvus hawaiiensis; Hawaiian crow). As the only remaining native crow species in Hawai?i, the ?Alala survived solely in a captive-breeding program from 2002 until 2016, at which point a long-term reintroduction program was initiated. The high-quality genome assembly was generated to lay the foundation for both comparative genomics studies and the development of population-level genomic tools that will aid conservation and recovery efforts. We illustrate how the quality of this assembly places it amongst the very best avian genomes assembled to date, comparable to intensively studied model systems. We describe the genome architecture in terms of repetitive elements and runs of homozygosity, and we show that compared with more outbred species, the ?Alala genome is substantially more homozygous. We also provide annotations for a subset of immunity genes that are likely to be important in conservation management, and we discuss how this genome is currently being used as a roadmap for downstream conservation applications.

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July 19, 2019

Extensive intraspecific gene order and gene structural variations between Mo17 and other maize genomes.

Maize is an important crop with a high level of genome diversity and heterosis. The genome sequence of a typical female line, B73, was previously released. Here, we report a de novo genome assembly of a corresponding male representative line, Mo17. More than 96.4% of the 2,183?Mb assembled genome can be accounted for by 362 scaffolds in ten pseudochromosomes with 38,620 annotated protein-coding genes. Comparative analysis revealed large gene-order and gene structural variations: approximately 10% of the annotated genes were mutually nonsyntenic, and more than 20% of the predicted genes had either large-effect mutations or large structural variations, which might cause considerable protein divergence between the two inbred lines. Our study provides a high-quality reference-genome sequence of an important maize germplasm, and the intraspecific gene order and gene structural variations identified should have implications for heterosis and genome evolution.

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July 19, 2019

High-quality genome assemblies reveal long non-coding RNAs expressed in ant brains.

Ants are an emerging model system for neuroepigenetics, as embryos with virtually identical genomes develop into different adult castes that display diverse physiology, morphology, and behavior. Although a number of ant genomes have been sequenced to date, their draft quality is an obstacle to sophisticated analyses of epigenetic gene regulation. We reassembled de novo high-quality genomes for two ant species, Camponotus floridanus and Harpegnathos saltator. Using long reads enabled us to span large repetitive regions and improve genome contiguity, leading to comprehensive and accurate protein-coding annotations that facilitated the identification of a Gp-9-like gene as differentially expressed in Harpegnathos castes. The new assemblies also enabled us to annotate long non-coding RNAs in ants, revealing caste-, brain-, and developmental-stage-specific long non-coding RNAs (lncRNAs) in Harpegnathos. These upgraded genomes, along with the new gene annotations, will aid future efforts to identify epigenetic mechanisms of phenotypic and behavioral plasticity in ants. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

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July 19, 2019

Deep genome annotation of the opportunistic human pathogen Streptococcus pneumoniae D39.

A precise understanding of the genomic organization into transcriptional units and their regulation is essential for our comprehension of opportunistic human pathogens and how they cause disease. Using single-molecule real-time (PacBio) sequencing we unambiguously determined the genome sequence of Streptococcus pneumoniae strain D39 and revealed several inversions previously undetected by short-read sequencing. Significantly, a chromosomal inversion results in antigenic variation of PhtD, an important surface-exposed virulence factor. We generated a new genome annotation using automated tools, followed by manual curation, reflecting the current knowledge in the field. By combining sequence-driven terminator prediction, deep paired-end transcriptome sequencing and enrichment of primary transcripts by Cappable-Seq, we mapped 1015 transcriptional start sites and 748 termination sites. We show that the pneumococcal transcriptional landscape is complex and includes many secondary, antisense and internal promoters. Using this new genomic map, we identified several new small RNAs (sRNAs), RNA switches (including sixteen previously misidentified as sRNAs), and antisense RNAs. In total, we annotated 89 new protein-encoding genes, 34 sRNAs and 165 pseudogenes, bringing the S. pneumoniae D39 repertoire to 2146 genetic elements. We report operon structures and observed that 9% of operons are leaderless. The genome data are accessible in an online resource called PneumoBrowse (https://veeninglab.com/pneumobrowse) providing one of the most complete inventories of a bacterial genome to date. PneumoBrowse will accelerate pneumococcal research and the development of new prevention and treatment strategies.

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July 19, 2019

A near complete, chromosome-scale assembly of the black raspberry (Rubus occidentalis) genome.

The fragmented nature of most draft plant genomes has hindered downstream gene discovery, trait mapping for breeding, and other functional genomics applications. There is a pressing need to improve or finish draft plant genome assemblies.Here, we present a chromosome-scale assembly of the black raspberry genome using single-molecule real-time Pacific Biosciences sequencing and high-throughput chromatin conformation capture (Hi-C) genome scaffolding. The updated V3 assembly has a contig N50 of 5.1 Mb, representing an ~200-fold improvement over the previous Illumina-based version. Each of the 235 contigs was anchored and oriented into seven chromosomes, correcting several major misassemblies. Black raspberry V3 contains 47 Mb of new sequences including large pericentromeric regions and thousands of previously unannotated protein-coding genes. Among the new genes are hundreds of expanded tandem gene arrays that were collapsed in the Illumina-based assembly. Detailed comparative genomics with the high-quality V4 woodland strawberry genome (Fragaria vesca) revealed near-perfect 1:1 synteny with dramatic divergence in tandem gene array composition. Lineage-specific tandem gene arrays in black raspberry are related to agronomic traits such as disease resistance and secondary metabolite biosynthesis.The improved resolution of tandem gene arrays highlights the need to reassemble these highly complex and biologically important regions in draft plant genomes. The updated, high-quality black raspberry reference genome will be useful for comparative genomics across the horticulturally important Rosaceae family and enable the development of marker assisted breeding in Rubus.

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July 19, 2019

How well can we create phased, diploid, human genomes?: An assessment of FALCON-Unzip phasing using a human trio

Long read sequencing technology has allowed researchers to create de novo assemblies with impressive continuity[1,2]. This advancement has dramatically increased the number of reference genomes available and hints at the possibility of a future where personal genomes are assembled rather than resequenced. In 2016 Pacific Biosciences released the FALCON-Unzip framework, which can provide long, phased haplotype contigs from de novo assemblies. This phased genome algorithm enhances the accuracy of highly heterozygous organisms and allows researchers to explore questions that require haplotype information such as allele-specific expression and regulation. However, validation of this technique has been limited to small genomes or inbred individuals[3]. As a roadmap to personal genome assembly and phasing, we assess the phasing accuracy of FALCON-Unzip in humans using publicly available data for the Ashkenazi trio from the Genome in a Bottle Consortium[4]. To assess the accuracy of the Unzip algorithm, we assembled the genome of the son using FALCON and FALCON Unzip, genotyped publicly available short read data for the mother and the father, and observed the inheritance pattern of the parental SNPs along the phased genome of the son. We found that 72.8% of haplotype contigs share SNPs with only one parent suggesting that these contigs are correctly phased. Most mis-phased SNPs are random but present in high frequency toward the end of haplotype contigs. Approximately 20.7% of mis-phased haplotype contigs contain clusters of mis-phased SNPs, suggesting that haplotypes were mis-joined by FALCON-Unzip. Mis-joined boundaries in those contigs are located in areas of low SNP density. This research demonstrates that the FALCON-Unzip algorithm can be used to create long and accurate haplotypes for humans and identifies problematic regions that could benefit in future improvement.

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July 19, 2019

Characterization of a human-specific tandem repeat associated with bipolar disorder and schizophrenia.

Bipolar disorder (BD) and schizophrenia (SCZ) are highly heritable diseases that affect more than 3% of individuals worldwide. Genome-wide association studies have strongly and repeatedly linked risk for both of these neuropsychiatric diseases to a 100 kb interval in the third intron of the human calcium channel gene CACNA1C. However, the causative mutation is not yet known. We have identified a human-specific tandem repeat in this region that is composed of 30 bp units, often repeated hundreds of times. This large tandem repeat is unstable using standard polymerase chain reaction and bacterial cloning techniques, which may have resulted in its incorrect size in the human reference genome. The large 30-mer repeat region is polymorphic in both size and sequence in human populations. Particular sequence variants of the 30-mer are associated with risk status at several flanking single-nucleotide polymorphisms in the third intron of CACNA1C that have previously been linked to BD and SCZ. The tandem repeat arrays function as enhancers that increase reporter gene expression in a human neural progenitor cell line. Different human arrays vary in the magnitude of enhancer activity, and the 30-mer arrays associated with increased psychiatric disease risk status have decreased enhancer activity. Changes in the structure and sequence of these arrays likely contribute to changes in CACNA1C function during human evolution and may modulate neuropsychiatric disease risk in modern human populations. Copyright © 2018. Published by Elsevier Inc.

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July 19, 2019

Degradation and remobilization of endogenous retroviruses by recombination during the earliest stages of a germ-line invasion.

Endogenous retroviruses (ERVs) are proviral sequences that result from colonization of the host germ line by exogenous retroviruses. The majority of ERVs represent defective retroviral copies. However, for most ERVs, endogenization occurred millions of years ago, obscuring the stages by which ERVs become defective and the changes in both virus and host important to the process. The koala retrovirus, KoRV, only recently began invading the germ line of the koala (Phascolarctos cinereus), permitting analysis of retroviral endogenization on a prospective basis. Here, we report that recombination with host genomic elements disrupts retroviruses during the earliest stages of germ-line invasion. One type of recombinant, designated recKoRV1, was formed by recombination of KoRV with an older degraded retroelement. Many genomic copies of recKoRV1 were detected across koalas. The prevalence of recKoRV1 was higher in northern than in southern Australian koalas, as is the case for KoRV, with differences in recKoRV1 prevalence, but not KoRV prevalence, between inland and coastal New South Wales. At least 15 additional different recombination events between KoRV and the older endogenous retroelement generated distinct recKoRVs with different geographic distributions. All of the identified recombinant viruses appear to have arisen independently and have highly disrupted ORFs, which suggests that recombination with existing degraded endogenous retroelements may be a means by which replication-competent ERVs that enter the germ line are degraded. Copyright © 2018 the Author(s). Published by PNAS.

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